Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. Field of the Invention
This invention relates to pluripotent stem cells and methods for isolating more committed progenitor cells from the pluripotent stem cells.
2. Background Art
Stem cells are undifferentiated, or immature cells that are capable of giving rise to multiple, specialized cell types and ultimately to terminally differentiated cells. These terminally differentiated cells comprise the fully functional organs and tissues within the adult animal and are the end product of embryonic development. Stem cells have two main characteristics. First, unlike any other cells, they are capable of dividing and differentiating into many different mature cell types within the body. Second, they are also able to renew themselves so that an essentially endless supply of mature cell types can be generated when needed. Because of this capacity for self-renewal, stem cells are therapeutically useful for the regeneration and repair of tissues. In contrast, terminally differentiated cells are not capable of self-renewal and are thus not capable of supporting regeneration and repair of damaged or diseased tissue.
The potency of a stem cell is measured by the number of different cell types it can ultimately produce. The most potent stem cell is the pluripotent stem cell (PSC) which can give rise to all cell types of the body (Wagner, E.; Matsui et at.; Resnick et al.). Other stem cells exist and include multipotent stem cells which give rise to two or more different cell types. For example, the multipotent hematopoietic stem cell is capable of giving rise to all cell types of the blood system (Jones et al.; Fleming et al.). Other known multipotent stem cells include a neuronal stem cell and a neural crest stem cell (Reynolds and Weiss; Stemple and Anderson). Bipotential stem cells are also considered multipotent stem cells since they give rise to more than one cell type. Specific examples of bipotential stem cells include the O-2A progenitor (Lillien and Raff; McKay, R.; Wolswijk and Noble) and the sympathoadrenal stem cell (Patterson, P. H.). There is one example of a monopotent stem cell, the epidermal stem cell (Jones and Watt).
The usefulness of stem cells for tissue regeneration and repair has been shown in several systems. For example, grafting of the hematopoietic stem cell has been shown to rescue an animal which has had its bone marrow subjected to lethal doses of radiation (Jones et al., supra). The O-2A progenitor has also been shown to remyelinate spinal cord neurons that have been chemically demyelinated (Groves et at.).
However beneficial these specific stem cells are, they still exhibit several practical drawbacks which limit their commercial development for biomedical applications. One disadvantage is their limited potency for developing into a broad range of cell lineages and tissues. Only the hematopoietic stem cell is capable of producing most cells within a tissue lineage, the other exhibit a very narrow range of developmental potential. Another disadvantage is the origin of the source material. Most neuronal stem cells have been isolated from newborn or early stage fetal tissue. The limited potency of these stem cells requires the independent isolation and maintenance of each cell type which is to be used for a specific application. Thus, the isolation, characterization and commercial usefulness of stem cells with other potentials will depend on the availability of large amounts of source material.
With the availability of a pluripotent stem cell, these disadvantages can be overcome if the pluripotent cell can be differentiated into more committed stem and progenitor cells. Differentiation into a stem cell with a desired potency and lineage specificity would allow an unlimited supply of source material and would also allow the treatment of a broad range of diseases due to the pluripotent nature of the stem cell. Such directed differentiation into a desired cell lineage would be very efficient and extremely cost effective for the commercial development of cellular therapeutics. However, because there are numerous differentiation pathways and points of commitment, and because the inductive effects are very complicated in the developing embryo, such directed differentiation of the pluripotent stem cell has not been accomplished in vitro.
To overcome the above limitations, those skilled in the art have resorted to time consuming experimentation or indirect methodologies in order to understand stem cell differentiation pathways and to isolate, through a series of in vitro and in vivo manipulations, more committed progenitor cells. For example, one of the most characterized stem cells is the bipotential 0-2A progenitor. It has been known for many years that this stem cell is capable of differentiating in vitro into either oligodendrocytes or type-2 astrocytes. However, it was only in recent years that the combination of growth factors needed to direct the differentiation down either pathway was fully understood. The sympathoadrenal stem cell is another such example where time consuming experimentation was necessary in order to understand its differentiation pathway. Although the differentiation pathways of these two bipotential stem cells are the most well characterized, the fact that it still took many years to understand their pathway exemplifies the problem of directed differentiation in vitro to obtain more committed progenitor or terminally differentiated cells.
There are also examples of the in vitro differentiation of multipotent and pluripotent stem cells. ES cells derived from blastocyst and post-implantation embryos can be allowed to uncontrollably differentiate into aggregates and embryoid bodies of terminally differentiated cells. Terminally differentiated cells within the aggregates and embryoid bodies comprise various cell types including extraembryonic endoderm, spontaneously contracting muscle, nerve and endothelial and fibroblast-like cells. ES cells can also be allowed to differentiate into cultures containing either neurons or skeletal muscle (Dinsmore et al.), or hematopoietic progenitors (Keller et al.; Biesecker and Emerson; Snodgrass et al.; Schmitt et al.). However, in none of these examples is the differentiation of the pluripotent stem cell directed down a particular pathway. Instead, they are allowed to differentiate randomly into a mixed population of terminally differentiated cells. Thus, there is no means of isolating a substantially pure population of progenitor cells of a desired cell lineage.
In order to obtain specific cell lineages differentiated from the pluripotent stem cell, those skilled in the art have relied on in vivo mechanisms to direct the differentiation into specific cell lineages. For example, (Otl et at., 1994), have described a method for isolating stem cells of the neuronal lineage after modifying pluripotent stem cells with a reporter construct and then reintroducing them into an early stage embryo. The reporter construct is expressed during neurogenesis and cells expressing the reporter gene are dissected out and placed in culture. Through in vivo mechanisms, this method allows for the isolation of cells committed to the neuronal lineage but, again, the dissected cells once placed in culture proceed to terminal differentiation.
Thus, there exists a need for a rapid method to differentiate and isolate more committed progenitor cells directly from stem cell cultures in vitro without undue experimentation. The present invention satisfies this need and provides related advantages as well.